Isoelectric focusing apparatus

ABSTRACT

Described is an isoelectric focusing electrophoretic process for the separation and purification of an amphoteric or neutral chemical compound from one or more electrically charged chemical compound(s). Said process is illustrated below at the example of the purification of a protein from contaminating proteins and salts. It may be carried out in an apparatus especially designed therefor, e.g. an apparatus as depicted in FIG. 1. Said apparatus and various modifications thereof are also claimed. The mixture to be separated is present within a hydraulic flow in chamber 8. Cylinders 5 and 12 contain immobilized pH-gradients or are replaced by amphoteric isoelectric pH-membranes. Each of said pH-gradients and pH-membranes has conductivity and both buffering and titrant capacity in its pH-interval. The extremities of said gradients or pH-membranes forming the ceiling and the floor of chamber 8 have isoelectric points equal to or just higher and just lower than the isoelectric point of the protein of interest which is kept at its isoelectric point in the hydraulic flow and does not enter said pH-gradients and pH-membranes. Contrary thereto the contaminating proteins and salts are driven by an electric field into said pH-gradients or via said pH-gradients or pH-membranes into the electrolyte reservoirs 3 and 14. The described process has the advantage that the desired compound need not be detected and extracted from any matrix, e.g. from said pH-gradients, and that the recovery and purity of the desired compound is higher.

This is a continuation of application Ser. No. 179,619 filed on Apr. 8,1988 now U.S. Pat. No. 4,971,670.

The present invention relates to a new and inventive process forseparating chemical compounds, e.g. peptides and proteins, having a zeronet electrical charge or being neutral under the experimental conditionsused, from other amphoteric or non-amphoteric chemical compounds, e.g.other peptides, proteins and/or salts, having a net electrical chargeunder said same experimental conditions by electrophoresis, especiallypreparative isoelectric focusing, and to a new means, i.e. an apparatus,for carrying out said process.

Preparative electrophoresis is a known technique and various forms ofelectrophoresis apparatus have been proposed for both analytical andpreparative purposes. Basically, the instrumentations and principles forpreparative electrophoresis can be classified into four main classes,according to the electrophoretic principle utilized [cf. A. T. Andrews,Electrophoresis: Theory, Techniques, and Biochemical and ClinicalApplications, Clarendon Press, Oxford 1986]:

a) disc electrophoresis

b) free curtain electrophoresis

c) isotachophoresis and

d) isoelectric focusing [cf. P. G. Righetti, Isoelectric Focusing:Theory, Methodology and Applications, Elsevier, Amsterdam, pp. 204-207(1983)].

In general, disc electrophoresis and isotachophoresis are run inhydrophilic matrices, either continuous (agarose and polyacrylamide) ordiscontinuous (granulated beds, such as Sephadex®). They arecharacterized by a high resolving power, but low tolerated sample loads.Free curtain electrophoresis in general utilizes continuous buffers, isperformed in a free liquid phase and is characterized by a continuouslyflowing thin film of buffer with a continuous sample input. Basicallythis technique offers large sample handling capacities but lowresolution. In addition, due to the higher diffusion coefficient ofproteins, this method is mostly confined to purification of intact cellsor subcellular organelles.

Isoelectric focusing (IEF) can be performed either in liquid supports(density gradients) or in gel media, either continuous or granulated. Infact, the technique of IEF was initiated as a preparative methodology,utilizing vertical glass columns filled with a sucrose density gradient.Moderately high sample loads could be handled with a high resolvingpower (ΔpI=0.02 of a pH unit; pI=isoelectric point), which was, however,severely lost when emptying the column via the bottom harvesting funnel.This technique has in fact today been essentially abandoned in favor ofIEF in gelatinous supporting media (mostly agarose and polyacrylamidematrices). The latter allows a high resolving power, but only moderateprotein loads. In addition, all preparative techniques which utilize asanticonvective media hydrophilic gels have the problem of recovering thepurified protein from the matrix. This requires additional handlingsteps, e.g. detection of the zone of interest, band cutting and elutionby diffusion or electrophoretic recovery. That has two majordisadvantages: a) low recoveries, as any matrix tends to irreversiblyadsorb proteins; and b) the possibility of contamination from gelmaterial (especially in the case of synthetic supports, such aspolyacrylamide, contamination from unreacted monomers and from short,oligomeric polyacrylamide coils non-covalently grafted to the bulkmatrix).

The present invention is based on the task to provide a process for thepurification of chemical compounds having, as peptides do, anisoelectric point or being uncharged under the conditions used, byelectrophoresis, wherein, contrary to the desired product, only theundesired by-products and contaminants come into contact with the matrixand which process gives excellent yields of the desired product in avery pure form.

Both the conception of said task itself and its solution involveinventive steps.

In the following, the invention will be described with reference to theaccompanying drawings of which:

FIG. 1 is a schematic over all view of an apparatus which can be used tocarry out the electrophoretic process according to the presentinvention,

FIG. 2 is a schematic exploded view of parts of an apparatus as depictedin FIG. 1,

FIG. 3 is a schematic exploded view of an alternative embodiment of anapparatus according to the present invention,

FIG. 4 is a schematic view of the most essential parts of anotheralternative embodiment of an apparatus which can be used to carry outthe process according to the present invention,

FIG. 5, FIG. 6 and FIG. 7 are diagrams illustrating the success ofelectrophoretic separations according to the process of the presentinvention,

FIG. 8 is a sectional view of a third alternative embodiment of anapparatus according to the present invention,

FIG. 9 is a view from below at a perforated disque which is a part ofthe apparatus depicted in FIG. 12,

FIG. 10 is a view from above at said disque,

FIG. 11 is a cross-sectional view taken along line XI--XI of FIG. 9, and

FIG. 12 is a cross-sectional view of a fourth alternative embodiment ofan apparatus according to the present invention.

The present invention relates to an isoelectric focusing electrophoreticprocess for the separation and purification of an amphoteric or neutralchemical compound, soluble in a solvent suitable for said process, fromone or more electrically charged chemical compound(s), soluble in saidsolvent, said process being carried out by using an electrophoreticapparatus, wherein the electric flow, i.e. the electric field, passingthrough the electrophoretic matrix, is coupled to a hydraulic flow 7, 8and 11 (cf. the figures), the direction of said electric flow beingdifferent from that of said hydraulic flow, said hydraulic flowcomprising a solution of said compound in said solvent and segmentingsaid matrix into two parts, one part, 5 or 25, being located at thecathodic side and the other, 12 or 26, being located at the anodic sideof said electric flow, characterized in that said amphoteric or neutralchemical compound is kept in an isoelectric or uncharged state withinthe hydraulic flow, and said charged chemical compound(s) is (are)removed from the hydraulic flow by the electric flow into at least oneof said parts of said matrix, or by way of at least one of said partsinto at least one of the electrolyte solution reservoirs 3 and 14, saidparts, independently of each other, representing immobilizedpH-gradients 5 and 12, each having conductivity and both buffering andtitrant capacity in its pH-interval, or amphoteric isoelectricimmobilized pH-membranes 25 and 26, each having conductivity and bothbuffering and titrant capacity at a specific pH-value.

The amphoteric or neutral compound, kept in an isoelectric or unchargedstate, is a chemical compound having no electrical net charge or beingneutral under the conditions of the purification process and at the timewhen the separation from the undesired accompanying chemical compound(s)actually takes place. It is preferably a protein, enzyme or smallerpeptide having at least two amino acids or a compound containing apeptide- or protein moiety, e.g. a glycoprotein, but also a nucleicacid, complex lipid or complex carbohydrate.

Contrary thereto, an electrically charged chemical compound is achemical species having an electrical charge under the conditions of thepurification process and at the time when the separation from thedesired chemical compound actually takes place, e.g. a protein, enzymeor smaller peptide being charged, i.e. non-isoelectric, and also a salt,e.g. an alkali metal salt, e.g. sodium chloride.

A solvent suitable for the process according to the present invention isany solvent solubilizing the desired chemical compound and allowing forthe necessary electric flow, e.g. water or a mixture of water with asuitable alcohol, e.g. a lower alkanol, for example methanol or ethanol,or an aqueous solution containing urea, detergents or any otherwater-miscible organic or protic solvent.

The electric flow is generated by the power supply 1. Any voltage thesystem can tolerate may be used, e.g. 100 to 10,000 volt, especially 500to 10,000 volt, preferably 500 to 5000 volt, e.g. 500, 1000, 5000 oreven 10,000 volt, provided the generated heat can be dissipated byproper cooling. At equilibrium, typical values are e.g. 1000 volt, 3 mAand 3 W or 500 volt, 10 mA and 5 W.

The electrophoretic matrix is a carrier for the electrophoreticseparation.

The hydraulic flow is generated e.g. by a pump, by stirring or byrotating the flow chamber 8 around a suitable axis and comprises asliquid phase a solution containing the mixture to be separated.

The direction of the hydraulic flow is at any suitable angle, e.g. 5° to90°, especially 30° to 90°, preferably about perpendicular (orthogonal),to the direction of the electric flow.

An immobilized pH-gradient as contained e.g. in cylinders 5 or 12comprises a stable pH-function on an electrophoretic matrix, e.g. a gel.Immobilized pH-gradients comprise a pH-interval which is generated in amanner known per se, e.g. by means of an overlayered density gradientand polymerization (cf. Application Note 321, dated August 1982, ofLKB-Produkter AB, Box 305, S-16126 Bromma, Sweden), e.g. by mixing equalvolumes of the two starting solutions A and B described below in agradient mixer, e.g. the "MicroGrad Gradient Maker" supplied byLKB-Produkter AB, the outlet of said mixer being connected with cylinder5 or 12. Starting solution A is an acidic, dense solution and containsbuffering Immobilines (registered trademark, used in the followingwithout indication) or an equivalent thereof, non-buffering Immobilinesor an equivalent thereof, Ampholines (registered trademark, used in thefollowing without indication) or an equivalent thereof, acrylamide,N,N'-methylene-bis-acrylamide, glycerol, water and suitablepolymerization catalysts. Starting solution B is a basic, light solutioncontaining buffering Immobilines, non-buffering Immobilines, Ampholines,acrylamide, N,N'-methylene-bis-acrylamide, water and suitablepolymerization catalysts, but no glycerol.

Amphoteric isoelectric immobilized pH-membranes are distinguished fromthe immobilized pH-gradients in that they do not comprise a pH-intervalbut have throughout the membrane the same pH-value. The manufacture ofthe membranes is similar to, but even simpler than the manufacture ofthe pH-gradients because no gradient mixer is required and no glycerolis necessary for preparing a density gradient.

The membranes are manufactured by polymerisation, preferably aroundneutral pH, at 50° C. in a forced-ventilation oven for 1 hour, of asolution of monomers (in general 10-15% T and 3-4% C) containingvariable amounts of buffering and titrant Immobilines in the ratiosneeded to generate the desired isoelectric point together withAmpholines, suitable polymerisation catalysts and water. It is essentialthat the membranes have a good buffering capacity at their isoelectricpoint in order to prevent electroendosmosis, a term denoting bulk liquidflow through the membrane caused by the presence or acquisition of a netelectrical charge. However, the Immobiline molarity should preferablynot exceed 50 mM of each Immobiline in the membrane.

Ampholines are low-molecular-weight amphoteric substances, i.e.ampholytes, which contrary to Immobilines are not fixed to theacrylamide/N,N'-methylene-bis-acrylamide polymer and are therefore ableto contribute to the electrical conductivity. Mixtures of manyamphoteric substances such as amino acids and peptides and someamphoteric and non-amphoteric buffer components can act as suitableampholytes. However, the great majority of iso-electric focusingexperiments are performed with the aid of commercial ampholyte mixtures.The most widely used of these, is marketed by LKB Produkter AB under thebrand name Ampholines. They consist of synthetic mixtures ofpolyaminopolycarboxylic acids with molecular weights mostly in theregion of 300-600. Other products can be used which contain sulphonic orphosphonic acid groupings in addition to the amino and carboxylic acidgroups. These products (Servalyts®, Serva-Feinbiochemica GmbH;Biolytes®, Bio-Rad Laboratories; Pharmalytes®, Pharmacia AB) haverecently been compared with the Ampholines and shown to have a similarperformance.

Immobilines are acrylamide derivatives with the general structure##STR1## where R contains either a carboxylic acid or a tertiary aminogroup. Immobilines are designed for co-polymerization with acrylamideand N,N'-methylene-bis-acrylamide in order to produce immobilizedpH-gradients. Each derivative has a defined and known pK-value.Acrylamide may be replaced e.g. by methacrylamide andN,N'-methylene-bis-acrylamide may be replaced by any other suitablecrosslinker, e.g. suitable other acrylamide derivatives.N-(3-Dimethylamino-propyl)-methacrylamide having a pK-value of 9.5 maybe mentioned as an example of a methacrylamide derivative beinganalogous to an Immobiline. After co-polymerisation the Immobilines arecovalently bound, i.e. immobilized, and do not contribute anything tothe conductivity of the pH-gradient or pH-membrane. However, theImmobilines contribute to the buffering and titrant capacity.

Preferably, the pH-gradients and pH-membranes are cast somewhere withina pH-range from about 3 to about 10, depending on the Immobilines andAmpholines available. If the compound of interest is amphoteric, thepH-values in the two gel extremities facing the flow chamber 8 have tobe set just above and below or equal to the isoelectric point of saidamphoteric substance with the precision required to keep it in theisoelectric state all the time. Said precision and the differencebetween the pH-values in said gel extremities, i.e. the width in termsof pH-units of the gap in between said gel extremities, depends on theresolving power needed, i.e. on the isoelectric points of thecontaminants which have to enter the gel, i.e. at least pass the gel. Inorder to achieve the highest possible resolving power, the pH-values insaid gel extremities can be the same and equal to the isoelectric pointof the desired compound. In that case it is advantageous to preventlosses of the desired compound which might occur by way of diffusion byinserting some appropriate mechanical means, e.g. a suitable milliporefilter, between gel and hydraulic flow. If the compound of interest isneutral, the pH-values in said gel extremities are chosen so that thecontaminants have to enter the gel. Said contaminants may stay withinthe gel or leave it again and accumulate in the anodic and cathodicchambers 14 and 3. Suitable polymerization catalysts are e.g.N,N,N',N'-tetramethylethylenediamine (TEMED) and ammonium persulphate.Said catalysts are added shortly before starting mixing the dense andlight solutions mentioned above. Other means for polymerization are e.g.riboflavin with ultraviolet light or gamma radiation.

In the gradient mixer, the basic, light solution is mixed into theacidic, dense solution which is simultaneously withdrawn into the outletof the gradient mixer which is connected to the container 5 or 12.Thereby the obtained density gradient co-varies with the pH-gradient.The lower end of containers 5 and 12 is provisionally sealed, e.g. withparafilm. After the polymerization process is finished, the parafilm isremoved. If the inside diameter of said container is too long it may benecessary to insert some support, e.g. a perforated plate, which is notremoved, at the lower end of the container. At least those parts of thecontainer coming in touch with the polymer have to be made of somematerial to which the polymer well adheres, e.g. of glass, in order toavoid the passage of some liquid between the wall of the container andthe polymer. The containers 5 and 12 containing the immobilizedpH-gradients are built into an apparatus according to the presentinvention, e.g. as shown in FIG. 1 and 2. Afterwards the gradients areproperly washed to remove undesired substances, e.g. unbound Immobilinechemicals, catalysts and ungrafted monomers. Otherwise, due to the verylow conductivity of the central portion of the gel as weak unboundanions and cations are electrophoretically depleted, the two saltfronts, accumulated towards the anodic and cathodic gel regions, arenever able to leave the gel. In order to achieve good focusing, theprimary, Immobiline gradient is overlayered with a secondary, carrierampholyte driven pH-gradient. The apparatus according to the presentinvention is usually run with the flow chamber full of liquid forseveral hours, e.g. about five hours, till attainment of steady-stateprior to sample application. Afterwards the flow chamber 8 and, ifnecessary, all other containers, coming in touch with the hydraulicflow, e.g. the sample reservoir 11, are emptied in order to removenoxious material leached out from the polymer, such as ungraftedmonomers, and filled with the sample to be purified.

During the entire process according to the present invention the samplesolution is vigorously stirred to prevent electrodecantation and kept atconstant temperature. The pH-gradients in containers 5 and 12 are alsokept at constant temperature. The temperature used depends inter alia onthe solvent, the stability and the solubility of the desired substance.In water, it is normally kept at a fixed value between about 1° and 20°C., e.g. at 2° C.

The basic concept of the present invention is that of a mixedpreparative technique, utilizing a liquid bed which may be a short oneand which is coupled to two gel phases delimiting it, and is illustratedin the following example of the purification of a protein. The proteinof interest is kept in an isoelectric state in the liquid phase, e.g. ina small, recycling chamber 8, while the impurities accompanying it aredriven away either towards the cathode 2 or the anode 13 and eventually(but not necessarily) focussed in the gel phases 5 and 12, representingthe pH-gradients (the numbers refer to the figures). The pH-gradientscan be replaced by pH-membranes. Thus, in this modified isoelectricfocusing technique, the protein of interest is not drivenelectrophoretically into the gel matrix (from which it would have to berecovered by an additional purification step), but is kept in anisoelectric state in the liquid stream (hydraulic flow) 7, 8 and 11constituting the sample feed and only the (electrically charged)impurities are forced to focus in the gel phases 5 and 12, delimitingthe liquid sample input, or to collect in one or both of the electrolytereservoirs 3 and 14. Preferably, the pH-value within the hydraulic flowcorresponds to the isoelectric point of the desired compound. Becausethe electrophoretic separation is performed in a pH gradient(isoelectric focusing), all the species having an isoelectric pointwithin the pH-gradient 5 or 12 are driven by the voltage gradient intothe particular zone where they exhibit zero net charge and in which theyremain stationary as long as the electric field is applied. Thedifference compared with previous techniques is inter alia that thestarting conditions are arranged in such a way that the component ofinterest is already isoelectric in the flow chamber 8 which constitutesthe sample feed of the system. Therefore, the component of interest isnot forced to migrate by the electric flow. Instead of using aconventional isoelectric focusing (IEF) system based on amphotericbuffers [cf. P. G. Righetti, Isoelectric Focusing: Theory, Methodologyand Applications, Elsevier, Amsterdam, pp. 204-207 (1983)], in theprocess according to the present invention a more advanced version ofit, an immobilized pH-gradient technique (IPG) [cf. P. G. Righetti, J.Chromatogr. 300, 165-223 (1984)] used.

A conventional isoelectric focusing (IEF) system would not be suitablefor the process according to the present invention for the followingreasons: a) IEF is not stable with time, in fact the pH gradient decaysand is subjected to a progressive acidification (cathodic drift) [cf. P.G. Righetti and J. W. Drysdale, Ann. N.Y. Acad. Sci. 209, 163-186 (1973)so that the protein of interest would not be kept in the liquid phase,but would eventually move into the gel matrix; b) due to the fact thatpH gradients are generated only in an approximate way in conventionalIEF systems, it would be impossible to set the boundary conditions inthe two gel extremities facing the flow-chamber 8 with the precisionrequired to keep the chemical compound of interest having an isoelectricpoint just in the isoelectric state all the time, thus preventing itfrom leaving the liquid phase [hydraulic flow; 7, 8 and 11]. In contrastthereto, with immobilized pH-gradients (IPGs) and pH-membranes, it is inmost cases possible to set the boundary conditions so that the anodicgel extremity facing the sample flow has a pH value just below theisoelectric point (pI) of the component of interest, while therespective cathodic gel extremity is set at a pH value just above the pIof the desired compound. Of course, the manufacture of suitable IPGs maybe difficult in the comparatively rare cases where the desired substancehas an extremely high or low pI. Said chemical compound, having anisoelectric point, will thus be isoelectric in this narrow pH gapdelimited by the two immobilized pH-gradients or pH-membranes. If thecompound of interest is amphoteric, this gap comprises normally 0.05 to0.2 pH-units; however, gaps comprising down to 0.001 pH-units can bealso achieved. It is also possible that the gap comprises 0 pH-units,i.e. the pH-values in said gel extremities correspond to the isoelecticpoint of the desired compound. This means that there is no pH-gap atall, but only a fluid gap between two gel phases. If the compound ofinterest is neutral, the pH-values in the gel extremities are not chosenin respect to the desired compound, but in respect to the undesiredamphoteric or charged compounds, in the sense that said undesiredcompounds should not have an isoelectric point within said pH-gap. Theneutral compound will never enter the pH-gradients, irrespective of theboundary conditions in the gel extremities facing the flow chamber 8. Inaddition to this precision in setting the boundary conditions, due tothe unlimited stability of IPGs with time, it is automatically ensuredthat the pH gradient never drifts so that the isoelectric conditions forthe chemical compound under purification will be constantly found in thehydraulic flow, especially in the flow-chamber 8, and not elsewhere,e.g. within the anodic or cathodic gel phases 12 and 5.

The process according to the present invention has at least thefollowing major advantages: a) extremely high sample recoveries,approaching 100%, as the chemical compound (e.g. the protein) underpurification never enters the gel phase, but is kept uncharged, e.g. inan isoelectric state, during the entire purification step in the liquidphase; b) large sample loads, as the compound to be purified, e.g. theprotein feed, may be kept circulating between a separate reservoir 11and the flow chamber 8 and only small amounts need be present at anygiven time in the electric field; c) a high resolving power, dependingon how narrow the pH interval selected across the isoelectric point (pI)of the desired compound, e.g. protein, is; d) automatic removal of anysalts or buffers accompanying the compound (e.g. the peptide or protein)of interest, which means that the present process can also be used forelectrodialysis (desalting process). Especially the removal ofmonovalent ions of strong acids or bases, e.g. Na.sup.⊕ and Cl.sup.⊖, isvery easy. For the removal of monovalent ions of weak acids and bases,e.g. ammonium and acetate, it is advantageous to use the amphotericisoelectric Immobiline membranes described below or rather shortpH-gradients, i.e. gradients comprising only a comparatively smallpH-range, e.g. 0.5 to 1.0 pH-units, substantially removed from thepK-values of the respective weak acids and bases. The removal ofmultivalent ions, e.g. sulphate, phosphate and citrate, takes more time,possibly due to the interaction of these species with the Immobilinematrix, and is best carried out under outside pH-control, e.g. with apH-stat, because otherwise, due to the faster removal of the monovalentcounterion, the solution in chamber 8 can become rather acidic oralkaline. Rapid desalting of protein samples for a variety of uses, e.g.enzyme reactions or ligand binding studies, is one of the problemscurrently faced in biochemistry.

Any salt content in the sample feed (already at 1 mM concentration)inhibits the transport of non-isoelectric proteins, perhaps because ofthe much larger current fraction carried by the ions themselves asopposed to proteins. In addition, high salt levels in the samplereservoir may form cathodic and anodic ion boundaries, alkaline andacidic, respectively, which may hamper protein migration and even inducedenaturation. In segmented (as well as in conventional) IPG gels,practically any level of salt present in the sample zone inhibits itselectrophoretic transport. Therefore, the best way to efficientlyeliminate protein impurities from an isoelectric component is tointroduce an already desalted protein feed into the segmented IPGapparatus. However, elimination of protein impurities can be achieved,although at a slower rate, even in the presence of salts in the sample.In the latter case, salt levels should be kept at the minimum compatiblewith protein solubility (e.g. 5 mM or lower) and external pH controlshould be exerted (e.g. with a pH-stat) so as to prevent drastic pHchanges in the sample feed, brought about by the generation ofboundaries produced by the salt constituents. In quite a few cases, aminimum salt concentration might be needed in the sample phase duringthe electrophoresis for preventing protein aggregation and precipitationdue to too low an ionic strength at or in the vicinity of theisoelectric point. For that purpose, an external hydraulic flow is used,replenishing the salt loss due to combined electric and diffusional masstransports (similar to the concept of Rilbes' steady-staterheoelectrolysis, H. Rilbe, J. Chromatography 159, 193-205[1978]).

The above-mentioned process may be carried out with one of the followingelectrophoretic apparatus belonging also to the subject of the presentinvention:

All of said electrophoretic apparatus basically comprise a flow chamber8 connected with two containers 5 and 12 each of which is filled,independently of the other, with an immobilized pH-gradient or replacedby an immobilized pH-membrane, one of which gradients or membraneshaving at its extremity connected with the flow chamber 8 an isoelectricpoint just below or equal to the isoelectric point of the chemicalcompound to be purified and being connected at its other extremity tothe anodic chamber 14 and the remaining pH-gradient or pH-membranehaving at its extremity connected with the flow chamber 8 an isoelectricpoint just above or equal to the isoelectric point of said chemicalcompound to be purified and being connected at its other extremity tothe cathodic chamber 3.

A schematic view of one of several possible modifications of this novelelectrophoretic apparatus is given in FIG. 1. A flow-chamber 8 isconnected to a sample reservoir 11 which, in principle, can hold anyvolume for processing via a pump 9 recirculating the feed through theelectric field. In general, the pump 9 is operated at maximum speed,e.g. 5 ml/min. Perpendicular to the hydraulic flow 7, an electric fieldis activated between two plates 2 and 13, preferably made of platinum,which serves to electrophoretically remove from the flow chamber 8 anyion or non-isoelectric amphoteric species. The flow-chamber 8 isconnected, e.g. via upper and lower O-ring seals 6 and 10, to twopolyacrylamide gel cylinders 5 and 12, held in short glass tubes, fittedwith jackets 19 for coolant flow 18 [19 and 18 are not shown in FIG. 1,but in FIG. 2]. The upper tube is connected, e.g. via a water-tightO-ring seal 4, to the cathodic chamber 3, containing in general adiluted base (e.g. 50 mM NaOH or ethanolamine, ethylendiamine, isoioniclysine or arginine), as is customary in conventional isoelectricfocusing (IEF). The lower tube 12 bathes its extremity directly in ananodic chamber 14, in general containing a diluted (strong or weak)acid, such as acetic acid, phosphoric acid or sulphuric acid or isoionicaspartic or glutamic acid solutions, just as routinely used in standardIEF. Obviously, the O-ring seals may be replaced by any other suitablemeans for connecting the various parts of the apparatus.

The novel feature of the present fractionation technique is that theflow-chamber 8 is delimited by the extremities of a lower and a upperpolyacrylamide gel representing immobilized pH-gradients orpH-membranes. Said pH-gradients are contained in the cylinders 12 and 5which are preferably made from glass or another suitable material towhich the gel is able to adhere by adhesive forces. By arranging theextremities of these two gel segments delimiting the flow-chamber 8 tohave isoelectric points (pI) just below (on the anodic side) or equal toand just above (on the cathodic side) or equal to the isoelectric pointof the desired compound, e.g. protein, under purification, this compoundwill in practice be titrated to its pI and as such will not be able toleave the hydraulic flow 8, 7 and 11. Conversely, all impurities havinga different pI, e.g. proteinaceous impurities, accompanying the compoundunder purification will automatically be [at the pH-value prevailing inthe flow chamber 8] either above or below their respective pIs, and thusbe forced to leave the chamber 8 and focus either in the lower or uppersegments 12 or 5 of the immobilized polyacrylamide gel or collect in theanodic or cathodic chambers 14 or 3. Given sufficient recycling timeunder a voltage gradient, all impurities leave the flow chamber 8 andthe pure compound, e.g. isoelectric protein, is recovered from the flowchamber 8 and the sample reservoir 11 originally containing the feed. Nofurther manipulations or sample extractions are needed, as the compound,e.g. protein, of interest stays all the time in the liquid phase anddoes not enter the gel.

The apparatus, which is assembled e.g. vertically or preferablyhorizontally, comprising the anodic and cathodic chambers 14 and 3, thegel cylinders 12 and 5, the seals 10, 6 and 4 and the flow chamber 8 isconnected to a power supply 1. At equilibrium, typical values are 1000V, 3 mA and 3 W, any other value being suitable for separations providedthe generated heat can be removed by proper cooling. The sampleflow-chamber 8 is provided by a means to keep it at a constanttemperature, and/or the feed is kept in a larger, jacketed reservoir 11,coupled to a thermostat 17. It is advantageous to keep the sample vessel11 under continuous, gentle stirring, otherwise, with time, a denserstratum could separate from a lighter one. Any pumping device 9, e.g. aperistaltic pump, is utilized for recycling, which is in generalperformed at maximum speed (e.g. 5 ml/min) so that the sample stays foras short a time as possible in the flow chamber 8, thus avoiding anyrisk of thermal denaturation. This is one of the simplest set-ups foroperation. In principle, any other probe or metering device can be builtaround this apparatus if needed: e.g. a biosensors detection system, animmunoelectrophoretic equipment, a laser excited fluorescence detectionequipment, any desired robotically coupled system, a device, e.g. aflowelectrode, for pH measurements and control, a device for radioisotopmonitoring and/or a device, e.g. a flow-cell for conductivitymonitoring, as needed. Obviously, for special purposes, the sample flow7 could also be monitored in the UV, or visible, or by fluorescentobservation, with the standard equipment coupled to chromatographiccolumns for following the rate of removal of some components in themixture.

FIG. 2 shows a schematic extended view of about the same apparatus asdepicted in FIG. 1 without showing the power supply 1, the stirrer 16and the thermostat 17 depicted in FIG. 1, but showing, in addition toFIG. 1, the jackets 19 for coolant flow 18 around the gel cylinders 5and 12, as well as the various components 3, 5, 6, 8, 10, 12 and 14 inthe correct position for assembly, but not yet assembled. The coolantflow 18 is connected to a thermostat, e.g. 17, not shown in FIG. 2.Although not shown in FIG. 2 (cf. however FIG. 1) the sample reservoir11 should also be kept at a constant temperature, e.g. 2° C., since theisoelectric points depend on the temperature. If desired, the flowchamber 8 may be also provided with jackets for coolant flow. FIG. 2, inaddition to FIG. 1, also shows a preferred form for the flow chamber 8:the inlet and the outlet for the hydraulic flow 7 are bent, one towardscylinder 12 and the other towards cylinder 5. Container 12, althoughdepicted with two screw-on-connections can be plunged directly into theanolyte solution in the electrode chamber 14.

FIG. 3 shows an apparatus suitable, after assembly of its components,for purifying two amphoteric compounds, e.g. two proteins, havingdifferent isoelectric points in the same apparatus and at the same time.Sample reservoirs 11a and 11b contain the initial feed which may be thesame or different. Sample reservoir 11a is connected via some kind oftubing 7a with one of two flow chambers 8a. Sample reservoir 11b isconnected via another tubing 7b with the second flow chamber 8b. Theflow chambers 8a and 8b are separated from each other by an intermediatecylinder 20 containing an immobilized pH-gradient. The extremity of saidintermediate pH-gradient directed to flow chamber 8b has a pH-value justhigher, e.g. +0.05 pH-units, than the isoelectric point of the desiredcompound in flow chamber 8b or the same pH-value as the desiredcompound. The extremity of said intermediate pH-gradient directed toflow-chamber 8a has a pH-value just lower, e.g. -0.05 pH-units, than theisoelectric point of the desired compound in flow chamber 8a or the samepH-value as the desired compound. At the end of the IEF process, thedesired purified species, e.g. proteins, are collected in chambers8a/11a and 8b/11b, any charged contaminants having been removed.

For analytical purposes, an apparatus according to FIG. 1 may be usedwherein, however, the flow chamber 8 is closed in as much as it is notconnected to the sample reservoir 11. In this case, the apparatus may bearranged in horizontal position, immersed into the same coolant androtated around its axis. Instead of rotating the entire apparatus, thesample in the flow chamber may be stirred, e.g. with a magnetic bar.

It is not only advantageous in the above-mentioned special case of aclosed flow chamber but also in the usual case of an "open" flowchamber, having in-and outlets for the hydraulic flow, to use theelectrofocusing apparatus in the horizontal position with said in- andoutlets in vertical position, the outlet being situated above the inlet.In the vertical arrangement, air bubbles tend to accumulate in the upperportion of the flow chamber. This results in an uneven transport ofimpurities and hindrance of the electric current flow. For the removalof the air bubbles, the apparatus has to be disassembled and positionedhorizontally to completely remove the bubbles through the outlet stream.Furthermore, the lower IPG segment, immersed in the lower electrolytereservoir (in general the anode), tends to swell. This forces the gel toprotrude from the supporting tube and eventually to detach from theglass walls and fall out of its lodging. These problems are eliminatedby a horizontal apparatus, e.g. as depicted in FIG. 8, provided withfilters 21 at all extremities of the IPG segments, for blocking theImmobiline gel phases in situ. The filters 21 are stretched in situ byan O-ring sitting on an annular ledge in the outer tube.

In FIGS. 1 to 3, the immobilized gel containers 5 and 12 are situatedopposite to each other. It is, however, also possible to arrange themparallel to each other as shown in FIG. 4. Such arrangement isespecially suitable for large scale purification since more than twocontainers 5 and 12, e.g. 4, 6 etc., may be immersed into the flowchamber 8.

For most purposes the process and the apparatus can be improved byreplacing at least one of the pH-gradients by amphoteric isoelectricimmobilized pH-membranes. Said membranes may be regarded as very shortpH-gradients covering only a very narrow pH-interval. Ideally, saidpH-interval comprises zero pH-units. Furthermore, the difference betweenthe pH-values in the extremities of the pH-gradients of pH-membranes,delimiting the flow chamber 8, may be also reduced to zero, i.e. theflow chamber may be delimited e.g. by two membranes having the samepH-value which is identical to the isoelectric point of the desiredcompound. This fact is surprising and facilitates the method in that twoidentical membranes can be prepared instead of membranes differing fromeach other. The use of pH-membranes instead of pH-gradients has theadditional advantage of being cheaper. Furthermore, the generated heatmay be removed more easily. Therefore, pH-membranes 25 and 26 will inmost cases be preferred when large scale purifications have to becarried out.

The invention relates also to an apparatus suitable for being used in anisoelectric focusing electrophoretic process as herein described, saidapparatus comprising a flow chamber 8 connected directly or indirectlyeither.

a) with two containers 5 and 12 each of which is suitable for beingfilled with and containing an immobilized pH-gradient, or

b) with two devices for taking up immobilized pH-membranes 25 and 26, or

c) with one container according to a) above and with one deviceaccording to b) above, one of which containers or devices beingconnected at its other extremity to the anodic chamber 14 and theremaining container or device being connected at its other extremity tothe cathodic chamber 3.

The following examples illustrate the invention without limiting it inany way.

ABBREVIATIONS

A: Ampere

C: (if used to describe the gel composition) percentage (by weight) oftotal monomer T (cf. below) which is due to the crosslinking agentN,N'-methylene-bis-acrylamide having the formula CH₂ ═CH--CO--NH--CH₂--CO--CH═CH₂

IPG: immobilized pH-gradient

pI: isoelectric point

T: total concentration [g/100 ml, i.e. weight per volume per cent] ofacrylamide and N,N'-methylene-bis-acrylamide

TEMED: N,N,N',N'-tetramethylethylenediamine

V: Volt

W: Watt.

EXAMPLE 1 Purification of a protein mixture

The experimental set-up is as in FIG. 1 and 2. The lower IPG segment 12having a total volume of 26 ml contains a pH 3.5-7.2 range (7% T, 4%C)-matrix and 1% carrier ampholytes in about the same pH interval and isprepared from an acidic dense solution and a basic light solution bymeans of a suitable gradient mixer as follows:

The acidic dense solution is prepared from a mixture of 685 μl of pK3.6, 223 μl of pK 4.6, 226 μl of pK 6.2, 118 μl of pK 7.0 and 154 μl ofpK 8.5 Immobilines (from stock 0.2M solutions), 0.6 ml Ampholine® pH3.5-7.0, 3.1 ml stock (30% T, 4% C)-acrylamide and 3.6 ml glycerol byadding water to 13 ml. The basic light solution is prepared from amixture of 124 μl of pK 3.6, 511 μl of pK 4.6, 347 μl of pK 6.2, 139 μlof pK 7.0, 310 μl of pK 8.5 and 238 μl of pK 9.3 Immobilines, 0.6 mlAmpholine pH 3.5-7.0 and 3.1 ml of stock (30% T, 4% C)-acrylamide byadding water to 13 ml. Once transferred to a suitabletwo-chamber-gradient-mixer, 10 μl of TEMED and 13 μl of 40% ammoniumpersulphate are added to each of the above-mentioned solutions.

The outlet of the gradient mixer is connected with chamber 12 the lowerend of which is provisionally closed, e.g. by parafilm, until thepolymerization process is finished. Polymerization proceeds for about 1hour at 50° C. (or for 2 hours at 37° C.).

The upper IPG segment 5 (26 ml total volume) contains a pH 7.4-10.0range, (7% T, 4% C)-matrix and 1% carrier ampholytes in the same pH-spanand is prepared from the below-mentioned solutions a) and b) as follows:

a) The acidic dense solution (pH 7.4) is prepared from a mixture of 506μl of pK 3.6, 387 μl of pK 7.0, 361 μl of pK 8.5 and 46 μl of pK 9.3Immobilines (from stock 0.2M solutions), 0.6 ml Ampholine pH 7-10, 3.1ml stock (30% T, 4% C)-acrylamide and 3.6 ml glycerol by adding water to13 ml.

b) The basic light solution (pH 10) is prepared from a mixture of 93 μlof pK 3.6, 335 μl of pK 7.0, 362 μl of pK 8.5 and 289 μl of pK 9.3Immobilines, 0.6 ml Ampholine pH 7-10 (all Ampholines from stock 40%solutions) and 3.1 ml of stock (30% T, 4% C)-acrylamide by adding waterto 13 ml.

Once transferred to the gradient mixer, solutions a) and b) are eachadded with catalysts (TEMED and ammonium persulphate, in this order) asabove.

The outlet of that gradient mixer is connected with chamber 5 the lowerend of which is provisionally closed.

After the polymerization is complete the means used for provisionallyclosing chambers 5 and 12 are removed and said chambers containing thethus prepared IPG-gels are built into the electrophoretic apparatusdepicted in FIG. 1. All non-amphoteric ions (ungrafted Immobilines,catalysts, buffers, etc.) are removed from the gel, prior to sampleapplication, by pre-running for 5 hours at 5 W/1000 Volt. Thus the flowchamber 8 is confined to a narrow pH interval (pH 7.2-7.4) centred onthe pI (7.30) of human adult hemoglobin (pI typical of human adulthemoglobin A [HbA] in an IPG gel at 10° C.). 70 mg total lystate from aheterozygous from human adult hemoglobin C [HbC] (containing ca. 60% HbAand 40% HbC), dissolved in 25 ml of 0.5% carrier ampholytes pH 6-8 arerecycled in the prefocused apparatus under 1000 Volt constant. At 30minute intervals 30 μl are sampled and kept at 4° C. for subsequentanalysis. The experiment is terminated with the last sampling after 23hours. The aliquots are analyzed in a (5% T, 4% C)-IPG gel in the pH6.5-8.5 span. The results obtained by densitometric scans of the peaksof HbA and HbC with a laser densitometer (provided by LKB) are presentedin FIG. 5 depicting along the x-axis the time [hours] and along they-axis the amount [mg] of HbA and HbC. Curve I (triangles) refers to HbAand curve II refers to HbC. It is seen that, while HbA stays constantfor the duration of the experiment, HbC is progressively removed till,at 23 hours, it cannot any longer be detected. After 12 hours ofrecycling, HbA is at least 95% pure while, after 23 hours, it is morethan 99.5% pure.

EXAMPLE 2 Removal of dyes from a protein

In order to evaluate the performance of the apparatus depicted in FIG. 1as an electrodialysis unit, the kinetics of removal of colored dyes (inthe form of salts) from protein mixtures are evaluated. In the anodicarm 12 an IPG pH 3.5-7.2 gel and in the cathodic arm 5 and IPG pH 7.4-10gel is polymerized. Thus the feed is kept at a pH between 7.2 and 7.4.The feed comprises a solution of 40 mg of purified human adulthemoglobin A in 0.5% Ampholine pH 6-8 added with 10 mg of an acidic dye(bromophenol blue) and with 10 mg of a basic dye (toluidine blue) (25 mltotal volume). The removal of said dyes subjected to 1000 V constant isfollowed by sampling 30 μl at given time intervals from the samplereservoir and assessing the residual amounts by spectrophotometricreadings at 600 nm. The results are shown in FIG. 6, depicting along thex-axis the time [hours] and along the y-axis the amounts [mg] of the twodyes. Curve I (triangles) shows the cathodic migration of toluidine blueand curve II refers to the anodic migration of bromophenol blue. Asshown in FIG. 6, after 2 hours essentially all of the dyes has beenremoved from the flow chamber, leaving behind the desalted hemoglobinsample. The rate of withdrawal seems to follow a first order reactionkinetic, as a plot (not shown) of log concentration vs. time is linear.The shape of the toluidine blue curve I is initially steeper than thatof bromophenol blue, but the measurements are complicated by the factthat this dye seems to consist of a family of three components, as threeblue zones were seen migrating in the upper gel.

EXAMPLE 3 Protein Desalting

30 ml of a solution of human adult hemoglobin A (HbA) are rendered 50 mMin NaCl and recycled in the apparatus depicted in FIG. 1 at 10 Wconstant and at 2° C. The recycling chamber is delimited by a pH 7.2floor and a pH 7.4 ceiling. The recycling speed is 10 ml/min. At thegiven time intervals, 2 ml aliquots are harvested, thermostated at 25°C. and monitored with an Analytical Control conductivity meter 101fitted with an Orion conductivity cell. The conductivity measurementsare converted into residual millimoles of NaCl. Desalting is essentiallycompleted in two hours. The kinetics of desalting of HbA are shown inFIG. 7 depicting along the x-axis the time [hours] and along the y-axisthe quantity [millimole] of sodium chloride.

EXAMPLE 4 Purification of N-acetyl-Eglin C

a) The isoelectric point of N-acetyl-Eglin C (pl=5.5) is determined onAmpholine PAG-plates pH 3.5-9.5, 5% T, 3% C, 2.2% Ampholineconcentration.

Ca. 350 μg total protein are applied in each pocket (in volumes up to 20μl) and then focusing is performed at 10 W limiting, 10 mA, and 1000 Vat equilibrium. The analytical runs are in general finished within 2hours and then the gels are fixed and stained with Coomassie Blue.

The fixing solution is prepared by dissolving 15 g trichloroacetic acidin double distilled water and adding double distilled water up to atotal volume of 100 ml.

The staining solution is prepared by dissolving 0.46 g of Coomassie BlueR 250 in 400 ml of the below-mentioned destaining solution. The obtainedsolution is heated to 60° C. and filtered before use. Theabove-mentioned destaining solution is prepared by adding doubledistilled water to 500 ml of ethanol up to a total volume of 1000 ml(solution I), by adding double distilled water to 80 ml of acetic acidup to a total volume of 1000 ml (solution II) and mixing solutions I andII in a ratio of 1:1 (v/v) before use.

b) For the manufacture of two amphoteric, isoelectric Immobilinemembranes, pH 5.5, 10.512 ml of a 0.2M solution of Immobiline pK 4.6 and9.664 ml of a 0.2M solution of Immobiline pK 9.3 are mixed and dilutedwith double distilled water to a total volume of 30.0 ml. The pH-valueof the solution thus obtained is determined by means of a pH-meter to be5.5. To said solution 40.0 ml of solution A (cf. below), 1.5 mlAmpholine pH 5-7, 96 μl TEMED, 120 μl of solution B (cf. below) anddouble-distilled water up to a total volume of 120.0 ml are added. Theabove-mentioned solution A is prepared by dissolving 28.8 g acrylamideand 1.2 g N,N'-methylene-bis-acrylamide in double distilled water andadding water up to a total volume of 100 ml. The above-mentionedsolution B is prepared by dissolving 400 mg ammonium persulphate in 880μl of double distilled water.

60.0 ml of the obtained solution are filled into each of two apparatusdescribed below (cf. c) and polymerised at 50° C. for one hour.

c) The apparatus mentioned above used for preparing the membranescomprises a plate made from an inert material, e.g.polytetrafluorethylene (Teflon®), which does not or only to a negligibledegree adhere to the polymerisate. On said plate a round perforateddisque (22) is placed which is separated from said plate by aring-shaped gasket (23) having a diameter of 9 cm and a height of 1 mm.FIG. 9 shows the view from below at said disque, FIG. 10 the view fromabove and FIG. 11 the cross sectional along view line XI--XI depicted inFIG. 9. The solution to be polymerised is filled through the holes 24 ofthe perforated disque.

d) The membranes obtained according to the procedure described abovetogether with the perforated carrier plates 22 are then built into acylinder having an inner diameter of about 9.5 cm and a height betweenthe membranes of about 3 cm. Said cylinder is fitted with an inlet 31and an outlet 30 opposite to each other for the hydraulic flow 7 and isused as flow chamber 8. If desired a millipore filter (8 μm) made fromcellulose-acetate or 6,6-polyamide (Nylon) or something like that, e.g.a polypropylene filter, may be placed between the pH-membranes 25 and 26and the hydraulic flow 7 preventing the substance to be purified (e.g.N-acetyl-Eglin C) from direct contact with the isoelectric membranes.The entire electrofocusing apparatus is assembled wherein theabove-mentioned cylinder with the built-in membranes replaces the flowchamber 8 and the immobilized pH-gradients 5 and 12. Preferably, saidcylinder is used in the horizontal position with the in-and outlet forthe hydraulic flow 7 in vertical position, the outlet being situatedabove the inlet. The advantage of said horizontal arrangement incomparison to the vertical assembly is that air bubbles arespontaneously removed.

FIG. 12 shows a cross-sectional view of the assembled apparatus. Acylindrical tube is segmented by the supported pH-membranes 22 into thecathodic chamber 3, the flow chamber 8 and the anodic chamber 14. Thecathode 2 and the anode 13 are connected via plugs 32 to the powersupply 1 [not shown]. The hydraulic flow 7 enters the flow chamber 8 viathe inlet 31 and leaves it via the outlet 30. The electrolyte solutionin the cathodic and anodic chambers may be renewed via the in- andoutlets 27 and 28. The various parts of the apparatus are held togetherby means of four thread poles 29 which are inserted through the holes33.

e) The assembled electrofocusing apparatus is prerun for 1 hour at 500volt, 25 mA and 10 W in a cold room (+5° C.) with the flow chamber fullof liquid but without the N-acetyl Eglin C-sample to be purified. Then,the flow chamber is emptied and filled with the sample to be purified.

f) 1 g of the sample containing recombinant DNA-N-acetyl Eglin C(purity: 80%, prepared according to European patent application no. 146785) is dissolved in 100 ml of 0.2% carrier ampholytes pH 5-7 andrecycled (20 ml/minute) in the prefocused apparatus under 500 Voltconstant and 10 mA/5 W in a cold room (+5° C.). At 30 minute intervals100 μl are sampled and kept at 4° C. for subsequent analysis. Theexperiment is terminated with the last sampling after 5 hours. Thealiquots are analyzed in an Ampholine PAG-plate pH 3-5 - 9-5, 5% T, 3%C, 2.2% Ampholine concentration. It is seen that all impurities areremoved after 3 hours of recycling.

If desired, the solutions in the cathodic 3 and anodic 14 chambers maybe pumped to a waste line at a speed of e.g. 5 ml/minute and regeneratedfrom big reservoirs.

EXAMPLE 5 Membranes Having Different Buffering Capacity

Analogs of the pl=5.50 membranes disclosed in Example 4 are prepared toincorporate a 10 mM, 40 mM or 100 mM concentration of the Immobilines.

While the 10 and the 40 mM "membranes" exhibit correct electroosmoticproperties and yield accurate experimental pI values, the 100 mM surfaceexhibits much larger dispersion and shows anomalous flow profiles in thepH range surrounding the pl. It seems thus reasonable to set an uppermolarity limit of about 50 mM of each Immobiline in the "membrane".

What is claimed is:
 1. An isoelectric focusing electrophoretic apparatussuitable for carrying out an isoelectric focusing electrophoreticprocess for the separation and purification of a desired amphotericchemical compound, soluble in a solvent suitable for said process, fromone or more amphoteric chemical compounds, soluble in said solvent, theisoelectric points of which differ from the isoelectric point of thedesired compound by at least 0.001 pH units, said process being carriedout by using an electrophoretic apparatus, wherein the electric flow,passing through the electrophoretic matrix, is coupled to a hydraulicflow, the direction of said electric flow being different from that ofsaid hydraulic flow, said hydraulic flow comprising a solution of saiddesired chemical compound in said solvent, said electrophoretic matrixbeing segmented into two parts by the hydraulic flow, said parts,independently of each other, representing immobilized pH-gradients (5)and (12), each having conductivity and both buffering and titrantcapacity in its pH-interval, or amphoteric isoelectric immobilized pHmembranes (25) and (26), each having conductivity and both buffering andtitrant capacity at a specific pH-value, one part (5) or (25), beinglocated at the cathodic side and the other, (12) or (26), being locatedat the anodic side, characterized in that said desired chemical compoundis kept in an isoelectric state within the hydraulic flow (7), (8) and(11), and the undesired amphoteric chemical compounds are removed fromthe hydraulic flow by the electric flow into at least one of the twoparts of said matrix, or through at least one of said parts into atleast one of the electrolyte solution reservoirs (3) and (14), saidapparatus comprising a flow chamber (8) connected directly or indirectlyeithera) with two containers (5) and (12) each of which is filled withan immobilized pH-gradient having conductivity and both buffering andtitrant capacity in its pH-interval, or b) with two amphotericisoelectric immobilized pH-membranes (25) and (26) having conductivityand both buffering and titrant capacity at a specific pH-value, or c)with one pH-gradient according to a) above and with one pH-membraneaccording to b) above, the isoelectric points in the extremities of thepH-gradients or membranes adjacent to the flow chamber (8) being equalto or just below the isoelectric point of the amphoteric chemicalcompound to be purified (anodic side) and equal to or just above theisoelectric point of said amphoteric chemical compound to be purified(cathodic side), one of which pH-gradients or pH-membranes beingconnected at its other extremity to the anodic chamber (14) and theremaining pH-gradient or pH-membrane being connected at its otherextremity to the cathodic chamber (3).
 2. An apparatus according toclaim 1 wherein the flow chamber (8) is connected via a pump (9) with asample reservoir (11).
 3. An apparatus according to claim 2 fitted witha device for keeping at least one of the pH-gradients, pH-membranes andthe sample at a constant temperature.
 4. An apparatus according to claim3 fitted by at least one of the following devices: a device forpH-measurements and control, a device for conductivity monitoring, abiosensors detection system, an immunoelectrophoretic equipment, a laserexcited fluorescence detection equipment, a robotically coupled system,a device for radioisotop monitoring or a device for monitoring thesample solution in the UV, or visible or by fluorescent observation. 5.An apparatus according to claim 3 fitted with a device for themechanical support of the pH-gradient gel in containers (5) and 12) orof the gel in the pH-membranes.
 6. An apparatus according to claim 3suitable for the simultaneous purification of two amphoteric or neutralcompounds comprising two separate flow chambers (8a) and (8b) separatedfrom each other by an intermediate immobilized pH-gradient (20) or oneor two pH-membranes.
 7. An apparatus according to claim 3 arranged insuch a position that air bubbles are removed from the flow chamber (8)by the hydraulic flow (7).
 8. An apparatus according to claim 7 whereinthe immobilized pH-gradients and pH-membranes have controlled bufferingand titrant capacity and contain ampholytes in an amount ensuringsufficient conductivity.
 9. An apparatus according to claim 3 whereinthe immobilized pH-gradients and pH-membranes have controlled bufferingand titrant capacity, pH-value and conductivity and can be prepared in areproducible manner.
 10. An apparatus according to claim 1 fitted within-and outlets (27) and (28) for renewing the electrolyte solutions inthe cathodic and anodic chambers (3) and (14).
 11. An apparatusaccording to claim 30 comprising two and not more than two amphotericisoelectric immobilized pH-membranes (25) and (26) each havingcontrolled buffering and titrant capacity at a specific pH-value.
 12. Anapparatus according to claim 1, suitable for the purification of apeptide, protein or compound containing a peptide or protein moiety,each of which having an isoelectric point between pH 5 and
 9. 13. Anapparatus according to claim 1, suitable for separation of amphotericchemical compounds the isoelectric points of which differ by at least0.05 pH-units.
 14. An apparatus according to claim 13 wherein thedirection of the hydraulic flow is orthogonal to the direction of theelectric flow.
 15. An apparatus according to claim 14 wherein thedirection of the hydraulic flow (7) is such that air bubbles are removedfrom the flow chamber (8).
 16. An apparatus according to claim 13wherein the immobilized pH-gradients have both buffering and titrantcapacity in their pH-interval and contain an amount of ampholytes in thesame pH-interval ensuring sufficient conductivity.
 17. An apparatusaccording to claim 13 wherein the immobilized pH-gradients andpH-membranes have controlled buffering and titrant capacity, pH-valueand conductivity and can be prepared in a reproducible manner.
 18. Anapparatus according to claim 17 wherein the desired compound is presentin an aqueous solution.
 19. An apparatus according to claim 1, whereinthe pH-value within the hydraulic flow corresponds to the isoelectricpoint of the desired compound.
 20. An isoelectric focusingelectrophoretic apparatus suitable for carrying out an isoelectricfocusing electrophoretic process for the separation and purification ofa desired amphoteric chemical compound, soluble in a solvent suitablefor said process, from one or more amphoteric chemical compounds,soluble in said solvent, the isoelectric points of which differ from theisoelectric point of the desired compound by at least 0.001 pH units,said process being carried out by using an electrophoretic apparatus,wherein the electric flow, passing through the electrophoretic matrix,is coupled to a hydraulic flow, the direction of said electric flowbeing different from that of said hydraulic flow, said hydraulic flowcomprising a solution of said desired chemical compound in said solvent,said electrophoretic matrix being segmented into two parts by thehydraulic flow, said parts, independently of each other, representingimmobilized pH-gradients (5) and (12), each having conductivity and bothbuffering and titrant capacity in its pH-interval, one part (5) beinglocated at the cathodic side and the other, (12) or (26), being locatedat the anodic side, characterized in that said desired chemical compoundis kept in an isoelectric state within the hydraulic flow (7), (8) and(11), and the undesired amphoteric chemical compounds are removed fromthe hydraulic flow by the electric flow into at least one of the twoparts of said matrix, or through at least one of said parts into atleast one of the electrolyte solution reservoirs (3) and (14), saidapparatus comprising a flow chamber (8) connected with two containers(5) and (12) each of which is filled with an immobilized pH-gradient,one of which gradients having at its extremity connected with the flowchamber (8) an isoelectric point just below the isoelectric point of theamphoteric chemical compound to be purified and being connected at itsother extremity to the anodic chamber (14) and the remaining pH-gradienthaving at its extremity connected with the flow chamber (8) anisoelectric point just above the isoelectric point of said amphotericchemical compound to be purified and being connected at its otherextremity to the cathodic chamber (3).
 21. An isoelectric focusingelectrophoretic apparatus suitable for being used in an isoelectricfocusing electrophoretic process for the separation and purification ofa desired amphoteric chemical compound, soluble in a solvent suitablefor said process, from one or more amphoteric chemical compounds,soluble in said solvent the isoelectric points of which differ from theisoelectric point of the desired compound by at least 0.001 pH units,said process being carried out by using an electrophoretic apparatus,wherein the electric flow, passing through the electrophoretic matrix,is coupled to a hydraulic flow, the direction of said electric flowbeing different from that of said hydraulic flow, said hydraulic flowcomprising a solution of said desired chemical compound in said solvent,said electrophoretic matrix being segmented into two parts by thehydraulic flow, said parts, independently of each other, representingamphoteric isoelectric immobilized pH membranes (25) and (26), eachhaving conductivity and both buffering and titrant capacity at aspecific pH-value, one part, (25), being located at the cathodic sideand the other, (26), being located at the anodic side, characterized inthat said desired chemical compound is kept in an isoelectric statewithin the hydraulic flow (7), (8) and (11), and the undesiredamphoteric chemical compounds are removed from the hydraulic flow by theelectric flow into at least one of the two parts of said matrix, orthrough at least one of said parts into at least one of the electrolytesolution reservoirs (3) and (14), said apparatus comprising a flowchamber (8) connected directly or indirectly with two devices for takingup immobilized pH-membranes (25) and (26), one of which is connected atits other extremity to the anodic chamber (14) and the remaining deviceis connected at its other extremity to the cathodic chamber (3).